Discovery of nigerose phosphorylase from Clostridium phytofermentans
Discovery of nigerose phosphorylase from Clostridium phytofermentans
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DOI：
10.1007/s00253-011-3515-9
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发表时间：
2012-02-01
影响因子：
5
通讯作者：
Kitaoka, Motomitsu
中科院分区：
文献类型：
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作者：
Nihira, Takanori;Nakai, Hiroyuki;Kitaoka, Motomitsu
对来自 Clostridium phytofermentans 的一种新型磷酸化酶进行了表征，该酶属于糖苷水解酶家族 (GH) 65 (Cphy1874)。大肠杆菌中产生的重组 Cphy1874 蛋白在无机磷酸盐存在的情况下对黑曲霉糖表现出磷酸解活性，导致 d-葡萄糖和 β-d-葡萄糖 1-磷酸 (β-G1P) 的释放，同时异头构型发生反转。黑曲霉糖磷酸解活性的动力学参数为k (cat) = 67 s(-1) 和K (m) = 1.7 mM。除了对曲二糖具有弱磷酸解活性外，该酶不会磷酸解典型 GH65 酶的底物，例如海藻糖、麦芽糖和海藻糖 6-磷酸。以d-葡萄糖为受体、β-G1P为供体的逆反应中表现出最高的逆磷酸解活性，反应初期产物主要为黑曲霉糖。该酶还显示出对 d-木糖、1,5-脱水-d-葡萄糖醇、d-半乳糖和甲基-α-d-葡萄糖苷的反向磷酸解活性（按递减顺序）。所有主要产物都是α-1,3-葡萄糖基二糖，尽管与d-木糖和甲基-α-d-葡萄糖苷的反应产生了大量的α-1,2-葡萄糖苷副产物。我们建议将 3-α-d-葡萄糖基-d-葡萄糖:磷酸 β-d-葡萄糖基转移酶作为该 Cphy1874 蛋白的系统名称，并将黑糖磷酸化酶作为该 Cphy1874 蛋白的简称。
A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and beta-d-glucose 1-phosphate (beta-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k (cat) = 67 s(-1) and K (m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and beta-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-alpha-d-glucoside. All major products were alpha-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-alpha-d-glucoside produced significant amounts of alpha-1,2-glucosides as by-products. We propose 3-alpha-d-glucosyl-d-glucose:phosphate beta-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.